a) Field of the Invention
The present invention relates to microscopes equipped for handling fluorescence applications.
b) Description of the Related Art
A known microscope for fluorescence applications is shown in FIG. 1. The beam path in a microscope equipped for fluorescence applications is shown in that figure.
The light from an additional light source (1) passes through a head-absorbing filter (2), red attenuating filter/stop slide (3) and a field diaphragm (4) to the excitation filter (5). The latter is installed in the reflector slide of the microscope which also contains a dichroic beam splitter (6). The dichroic beam splitter reflects the shortwave excitation light through the objective (7) into the specimen or preparation (8).
The occurring emission is collected by the objective (7) and—because it has greater wavelengths than the excitation light is—passed by the dichroic beam splitter (6). The beams now pass through the emission filter (9). The remainder of the excitation light is filtered out by the latter. For this reason, this filter is also referred to as a blocking filter. As is conventional, the tube lens (10) and eyepiece (11) form the microscope image formed of fluorescent light.
The Problem Addressed by the Invention
In order to avoid image offset (pixel shift), multiple exposures in fluorescence recordings with different emission filter sets (A, B) require an optimal congruence of the object image in the individual recordings. However, there are technological limits in this respect.
Because of the different wedge angles of the emission filters (AEM, BEM) and of the color splitters, the filter combinations needed for the fluorescence application cause a slight image offset. This is shown in FIG. 2.
The reference symbols have the following meanings:                AEm emission filter of filter set A        BEm emission filter of filter set B        a1 light beam striking AEm         b1 light beam striking BEm         a2 light beam deflected by AEm         b2 light beam deflected by BEm         αA angle between the incident light beam a1 and the deflected light beam ac of filter AEM         αB angle between the incident light beam b1 and the deflected light beam b2 of filter BEM         E image plane        {overscore (PAPB)} distance (pixel shift) between the image points impinging on the image plane E        
The light beams a1 and b1 impinge on the emission filters AEm and BEm of the corresponding filter sets A and B. The beam is deflected in more or less opposite directions because of the existing wedge angle of the filters depending on the installed position (a2 and b2 are greatly exaggerated in the drawing in order to illustrate the process). Therefore, the image points impinging on the image plane E do not lie exactly one above the other, but are offset relative to one another by the pixel shift. Even with the close tolerances of the filters sets by Carl Zeiss with a slight image offset, this offset still occurs to a slight extent.